Amphipathic copolymers such as poly(styrene-maleic acid) (SMA) are promising tools for the facile extraction of membrane proteins (MPs) into native nanodiscs. Here, we designed and synthesized a library of well-defined alternating copolymers of SMA analogues in order to elucidate polymer properties that are important for MP solubilization and stability. MP extraction efficiency was determined using KcsA from E.coli membranes and general solubilization efficiency was investigated via turbidimetry experiments on membranes of E.coli, yeast mitochondria and synthetic lipids. Remarkably, halogenation of SMA copolymers dramatically improved solubilization efficiency in all systems, while substituents on the copolymer backbone improved resistance to Ca2+. Relevant polymer properties were found to include hydrophobic balance, size and positioning of substituents, rigidity and electronic effects. The library thus contributes to the rational design of copolymers for the study of MPs.
The manuscript now includes more detailed investigation, i.e, dose-response curves, negative stain electron microscopy, and a summarizing heat-map to give a clear overview of structure-activity relationships and how these relate to various membrane systems. There are also minor modifications throughout to improve both grammar as well as overall clarity. Use of colour in several figures were also modified to improve legibility. Additional copolymers were synthesized, characterized and tested to evaluate the role of copolymer length, as well as the combination of several modifications into one copolymer (alpha-MeSAA). Finally, all of the copolymers have now been thoroughly characterized by both 1H as well as 13C NMR.
SI - SMA Analogues